Polysialic acid (PSA) is a developmental regulated, anti-adhesive glycan that is added to the neural cell adhesion molecule (NCAM), the most abundant of five mammalian polysialylated proteins. The presence of PSA on NCAM N-glycans negatively modulates cell adhesion and is critical for a variety of important processes including brain development, learning and memory, neuronal regeneration, and the growth and invasiveness of cancer cells. Our goal in this proposal is to understand the mechanism of protein-specific polysialylation and how the polysialyltransferases (polySTs) recognize NCAM. We have demonstrated that the first fibronectin type III repeat (FN1) of NCAM is required for the polysialylation of the N-glycans found on the adjacent immunoglobulin domain (Ig5). We have solved the crystal structure of FN1 and shown that an acidic surface patch is involved polyST recognition, and that a unique helix, which links the strands 4 and 5 of the FN1 beta sandwich structure, is critical for positioning the Ig5 N-glycans for polysialylation. We propose experiments to elucidate the mechanism of protein-specific polysialylation and to test the hypothesis that polyST-FN1 binding and an lg5-FN1 interaction are required for NCAM polysialylation.
In aim I we will identifiy the FN1 residues required for polyST recognition using gain- and loss-of-polysialylation experiments.
In aim II we will determine the FN1 sequences required for, and factors modulating, polyST-NCAM binding using co-immunoprecipitation and in vitro binding assays.
In aim III, we will obtain the crystal structure of lg5-FN1 to determine whether these domains interact, the role of the FN1 helix in this interaction, and use binding assays and rotary shadowing electron microscopy to evaluate an alternate possibilty that the polySTs allow a transient lg5-FN1 interaction during polysialylation. Our long term goal is to understand the basis for the protein specificity of polysialylation so that we can design approaches to eliminate or enhance NCAM polysialylation during development and disease. Relevance to public health: Polysialic acid (PSA) is anti-adhesive sugar that is added specifically to the neural cell adhesion molecule, NCAM. PSA is critical for brain development, neuronal regeneration, and promotes cancer invasiveness. The goal of this project is to understand how the enzymes that add PSA recognize NCAM so that we can design approaches to diminish or enhance NCAM polysialylation and regulate its effects on cell adhesion during development and disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM063843-08
Application #
7841764
Study Section
Intercellular Interactions (ICI)
Program Officer
Marino, Pamela
Project Start
2001-07-01
Project End
2012-03-31
Budget Start
2010-04-01
Budget End
2012-03-31
Support Year
8
Fiscal Year
2010
Total Cost
$287,727
Indirect Cost
Name
University of Illinois at Chicago
Department
Biochemistry
Type
Schools of Medicine
DUNS #
098987217
City
Chicago
State
IL
Country
United States
Zip Code
60612
Thompson, Matthew G; Foley, Deirdre A; Colley, Karen J (2013) The polysialyltransferases interact with sequences in two domains of the neural cell adhesion molecule to allow its polysialylation. J Biol Chem 288:7282-93
Zapater, Joseph L; Colley, Karen J (2012) Sequences prior to conserved catalytic motifs of polysialyltransferase ST8Sia IV are required for substrate recognition. J Biol Chem 287:6441-53
Thompson, Matthew G; Foley, Deirdre A; Swartzentruber, Kristin G et al. (2011) Sequences at the interface of the fifth immunoglobulin domain and first fibronectin type III repeat of the neural cell adhesion molecule are critical for its polysialylation. J Biol Chem 286:4525-34
Foley, Deirdre A; Swartzentruber, Kristin G; Thompson, Matthew G et al. (2010) Sequences from the first fibronectin type III repeat of the neural cell adhesion molecule allow O-glycan polysialylation of an adhesion molecule chimera. J Biol Chem 285:35056-67
Foley, Deirdre A; Swartzentruber, Kristin G; Lavie, Arnon et al. (2010) Structure and mutagenesis of neural cell adhesion molecule domains: evidence for flexibility in the placement of polysialic acid attachment sites. J Biol Chem 285:27360-71
Colley, Karen J (2010) Structural basis for the polysialylation of the neural cell adhesion molecule. Adv Exp Med Biol 663:111-26
Foley, Deirdre A; Swartzentruber, Kristin G; Colley, Karen J (2009) Identification of sequences in the polysialyltransferases ST8Sia II and ST8Sia IV that are required for the protein-specific polysialylation of the neural cell adhesion molecule, NCAM. J Biol Chem 284:15505-16
Mendiratta, Shalu Shiv; Sekulic, Nikolina; Hernandez-Guzman, Francisco G et al. (2006) A novel alpha-helix in the first fibronectin type III repeat of the neural cell adhesion molecule is critical for N-glycan polysialylation. J Biol Chem 281:36052-9